The major problems of current genetic engineering processes of plants can be summarized as follows:    1. Generally low yield of the product of interest. Examples are pharmaceutical proteins, enzymes, other proteins, polymers, sugars, polysaccharides, etc. Low yield means high purification/downstream processing costs.    2. Genetic contamination due to free crossing of genetically modified plants with non-transgenic varieties or wild relatives as well as presence of “volunteer” plants in the fields following the harvest. Such events easily occur spontaneously because plants are self-replicating organisms or because of human mistakes or deliberate actions.
Both problems are to a great degree flip sides of the same problem: low yield is usually a result of our desire to construct a plant that is a producer which at the same time retains an ability of going through a full development and reproduction cycle. Combining the process of development and reproduction with the process of transgene expression does not give a solution. Inducible (U.S. Pat. Nos. 5,187,287; 5,847,102; Mett et al., 1993, Proc. Natl. Acad. Sci., 90, 4567-4571; Aoyama & Chua, 1997, Plant J., 11, 605-612; McNellis et al., 1998, Plant J., 14, 247-257; U.S. Pat. No. 6,063,985; Caddick et al., 1997, Nature Biotech., 16, 177-180; WO09321334; Weinmann et al., 1994, Plant J., 5, 559-569) or organ-specific transgene expression systems (U.S. Pat. No. 5,955,361; WO09828431; De Jaeger et al., 2002, Nature Biotech., 20, 1265-1268) do also not provide a solution to the problem of genetic contamination and do not provide for high yield of the product of interest. All inducible and tissue-specific promoters used in the systems mentioned above have basal activity, e.g. they are “leaky”, which in the course of generations causes transgene silencing, thus inevitably decreasing the yield of the product of interest. Moreover, the yield is not the only problem, as transgenic plants carrying recombinant genes under control of such inducible systems do not fulfill stringent biosafety criteria, for example in the case of production of recombinant biopharmaceuticals.
The use of amplification vectors based on plant viral elements can potentially provide a partial solution to the mentioned problems, especially when such vectors are used for transient expression (U.S. Pat. Nos. 5,491,076; 5,977,438; 5,316,931; 5,589,367; 5,866,785; WO0229068). However, most of the vectors for transient expression experiments were developed for selected plant species, predominatly of the Nicotiana family (U.S. Pat. Nos. 5,466,788; 5,670,353; 5,866,785; WO02088369). The efficiency of viral vectors for the production of recombinant protein/RNA of interest is also determined by their ability for efficient cell-to cell and systemic movement. The latter parameters are species- and variety dependent, thus severely restricting the freedom to choose hosts for transient expression. A potential solution to this problem is the design of transgenic plants carrying the viral vector stably integrated into the chromosomal DNA. The use of viral vectors designed for the expression of foreign sequences in plants via transfection or stable incorporation into the plant chromosomal DNA was described in numerous reviews (Stanley, J., 1993, Curr Opin Genet Dev., 3, 91-96; Schlesinger, S. 1995, Mol Biotechnol., 3, 155-165; Porta, C. & Lomonossoff, G. 2002, Biotechnol. & Genel. Eng. Rev., 19, 245-291; Awram et al., 2002 Adv Virus Res., 58, 81-124). However, attempts to design transgenic plants with an amplicon vector stably integrated into the chromosomal DNA usually faces the problem of transgene silencing, thus turning this approach useless. In the best case, it provides a yield that is slightly higher than that provided by a strong constitutive promoter, as was shown in late 80s by Hayes and colleagues for GUS and NPT genes (Hayes et al., 1989, Nucl. Acids Res., 17, 2391-2403). Since that time there was no significant breakthrough in achieving high yield, biologically safe recombinant protein production in transgenic plants based on viral replicons.
The work of Mallory and colleagues (2002, Nature Biotechnol., 20, 622-625) on amplicons in hybrid plants, whereby said hybrid plants provide for a suppressor of post-transcriptional gene silencing (PTGS), offers a partial solution to the problem. However, this approach did not give a high yield of the product of interest, that is incompatible with normal plant development. Further, biosafety issues were not addressed. Gooding and colleagues (1999, Nucleic Acids Res., 27, 1709-1718) reported replication of geminiviral vectors in isolated wheat embryos in a scientific study. Possible technical applications were not addressed. Further, this method cannot be scaled up and is therefore of no use for technical applications.
It is therefore an object of the invention to provide a novel process of producing a product of interest like a protein of interest in a plant production system, notably in high yield. It is another object of the invention to provide a biologically safe process of producing a product of interest, notably a protein of interest, in a plant production system, whereby distribution in the environment of transgenic genetic material involved in said process is tightly controlled and occurs with low probability. It is a further object of the invenion to provide a process of producing a product of interest like a protein of interest in a plant production system, whereby plant growth and isolation of the product of interest can be decoupled in space and time without loosing yield or quality of said product of interest.